Field flow fractionation (FFF) is known in a variety of implementations to separate populations of macromolecules or nanoparticles (colloidal particles) in a range of sizes from about 0.5 nm to a few microns into subranges of particle size to enable more accurate determination of particle size distribution. FFF is described in more detail in U.S. Pat. No. 4,147,621, granted Apr. 3, 1979, entitled “Method and Apparatus for Flow Field-Flow Fractionation,” issued to John C. Giddings, and herein incorporated by reference. Many detection methods respond more readily to large particles than small (or vice versa) so improved quantification is possible if a sample that is mixed (heterogeneous) in size can be fractionated.
It is particularly convenient if this can be done continuously and FFF methods do this for a wider range of sizes in the range mentioned, and more rapidly and effectively than some other methods such as the use of a size exclusion chromatography column.
In general an FFF technique consists in flowing a suspension of particles axially through a small channel, which can be a fraction of a millimeter in depth, a few millimeters wide, and a few centimeters long, with a force applied at right angles to the flow. We will consider this to be in a vertical direction but the orientation is usually not important. The force can arise in a variety of ways, and we will consider cross flow (XF FFF, XF4) to be the method of choice, but gravitation and temperature among others have been used.
This right angle force tends to propel the sample particles to the bottom of the channel: in the case of XF4 this is effected by making the bottom of a membrane through which the solvent supporting the particles can penetrate, leaving the latter in the channel chamber itself. This tendency for particles to reside near the wall is countered by the natural Brownian motion (diffusion) of the particles and more readily in the case of the smaller sizes: hence as the suspension flows along the channel the smaller particles are less hindered by interaction with the membrane (wall) and tend to emerge (elute) before the larger ones. In practice, for a channel of a few centimeters in length, with a flow rate of 0.5 ml/minute, a sample of size 20 nanometers (nm) may emerge after ˜8 minutes while one of 100 nm may emerge after 20 minutes. An example of the elution of a trimodal mixture is shown in FIG. 7. The green (unbroken) trace plots the intensity of light scattered as a function of time or elution volume: the two are considered commensurate since the fluid is pumped at a constant rate. In parallel, a continuous measurement of size is performed using dynamic light scattering to record the z-average hydrodynamic diameter. If the sample composition is known, or only relative measurements are required, such an absolute detection method may not be needed, but it can be a valuable diagnostic.